Precision flow control system

ABSTRACT

A precision flow controller is capable of providing a flow rate less than 100 microliters/minute and varying the flow rate in a prescribed manner that is both predictable and reproducible where the accuracy and precision of the flowrate is less than 5% of the flow rate. A plurality of variable pressure fluid supplies pump fluid through a single outlet. Flowmeters measure the flow rates and a controller compares the flow rates to desired flowrates and, if necessary, adjusts the plurality of variable pressure fluid supplies so that the variable pressure fluid supplies pump fluid at the desired flow rate. The variable pressure fluid supplies can be pneumatically driven.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of the U.S. patentapplication Ser. No. 12/334,375 filed Dec. 12, 2008, now U.S. Pat. No.7,927,477 which is a divisional of U.S. patent application Ser. No.10/246,284, filed Sep. 17, 2002, now U.S. Pat. No. 7,465,382 that isrelated to U.S. patent application Ser. No. 10/155,474, filed May 24,2002, that is a continuation-in-part of U.S. patent application Ser. No.09/942,884 filed Aug. 29, 2001 that claims the benefit of U.S.Provisional application No. 60/298,147 filed Jun. 13, 2001, the entiredisclosures of which are incorporated by reference in their entirety forany and all purposes.

BACKGROUND

The invention pertains to the field of precision fluid control for HighPerformance Liquid Chromatography (“HPLC”) including gradient liquidchromatography (“LC”). Commercially available HPLC systems for carryingout analytical separations typically control fluid at flow rates of afew milliliters per minute (“ml/min”) for columns of 4-5 mm in diameter.The current trend in LC is reducing the size and flow rates of thesystem in order to reduce the amount of waste generated, lower samplesize requirements, and improve compatibility with robust detectionsystems such as mass spectrometers (“MS”). There is particular interestin columns with diameters in the range of 50 μm to 1 mm that aretypically referred to as capillary columns. Since the flow rate of fluidin these capillary systems can range from nanoliters per minute(“nL/min”) to 100 microliters per minute (“μl/min”), there is a need forprecision flow control at low flow rates.

The precise control of flow rates is essential to analysis using HPLC.Uncontrolled variations in the flow rate and the fluid composition inHPLC systems produces a number of deleterious effects that compromisethe utility and sensitivity of the method. Particularly, a known flowrate is required to reliably predict analyte retention times and thusreliably identify analytes. Also, temporal variations in flow rate, suchas pulsations, can produce variations in detector signal that may beconfused with the presence of an analyte, resulting in theidentification of false analyte features. Temporal variations in flowrate may produce fluctuations in the detector baseline obscuring traceanalyte signatures, degrading the minimum detectivity of the system.

The importance of flow control is even more critical for gradientseparations where the fluid composition is varied during the course ofthe separation. In gradient HPLC, the fluid outputs from multiple pumpsare summed to provide a desired flow rate of varying composition. Sincethe retention time of an analyte is very dependent on the time-varyingcomposition of the eluting fluid, precise control of all fluids iscritical.

Conventional HPLC pumping systems generally employ positive displacementmethods, where the rate of mechanical displacement of a pump element,e.g. a lead-screw driven piston, provides a proportional rate of liquidflow. This method scales down poorly to low flow rates and is unable tocontrol fluid flow with sufficient accuracy to generate reliable andrapid gradients for capillary HPLC systems. The origin of the low flowrate inaccuracies include: check valve leakage, pump seal leakage,flexing and creep of mechanical seals, thermal expansion of componentsand compression of the working fluid. Many of these issues can produceerrors in flow rate larger than the flow rates desired in capillarychromatography. These systems also typically include pulse dampenersand/or fluid volumes to dampen fluctuations due to piston refilling.These volumes produce relatively high hydraulic capacitance in thesystem. This capacitance, in conjunction with the high hydraulicresistance of microbore columns, leads to slow time response.

The most common approach to achieve flow rates compatible with capillaryHPLC is to split the pump output of conventional HPLC pumps. This methodis described in Johannes P. C. Vissers, “Recent developments inmicrocolumn liquid chromatography” Journal of Chromatography A, vol 856pp. 117-143 (1999); U.S. Pat. No. 6,402,946; and U.S. Pat. No.6,289,914. In the flow splitting approach, some portion of the mixedsolvent is split-off to provide the required flow rate to the column,the balance of the liquid is shunted to waste. The splitting elementemploys two different flow conductance paths to produce the two flowstreams. The precision of this method is limited by changes that mayoccur in the relative hydraulic conductances of the two flow paths overtime. Conductance changes, such as partial plugging of thechromatographic column, will result in changing the flow rate of fluidinto the separation column. An additional problem with flow splitting isthat it has proven difficult to remove delay times in gradients thatresult from dead volumes of the splitter based systems. Additionally,these systems generate large volumes of waste relative to the fluid thatis actually used since the splitter discards the majority of the fluidin the system.

Agilent Technologies has recently attempted to address one of theproblems of flowsplitters using a variable flow splitter with activefeedback as disclosed at www.agilent.com. In this capillary HPLC system,high pressure fluids for the gradient separation are delivered byconventional positive displacement pumps and mixed at high volumetricflow rates (˜1 ml/min). The flow rate of the mixture delivered to thecolumn is directly measured and maintained by actively controlling avariable splitter valve. The system is still limited by the delayvolumes (˜5 μl) which effects chromatographic performance and theability to accurately measure flow rates in a varying mixture. At flowrates typical for 300 and 100 μm diameter columns this introduces delaytimes of 1-10 minutes in the gradient. The delay times make flow ratemeasurements difficult since they depend on knowing the physicalproperties of the mixture in the flow sensor at a specific time. Despitethe effort to carefully control flow rates, changes in the conductanceof the capillary column can result in >20% errors in flow rate and take˜30 seconds for the system to respond to the conductance change.

In addition, there are several other HPLC systems that have active flowrate control. These systems were developed for high flow rate (>0.1ml/minute) isocratic HPLC. An early system was developed by DuPontaround 1970. The Dupont 833 precision Flow Controller worked togetherwith the Dupont 830 liquid chromatograph (H. M. McNair and C. D.Chandler “High Pressure Liquid Chromatography Equipment—II”, J. ofChrom. Sci., v12, pp 425-432 (1974)). The system worked by measuring thepressure drop across a flattened capillary downstream of the pump. Themeasured flow rate was used to modify the air pressure on a pneumaticpiston. The system was designed to work with column diameters of 2.1 to23.5 mm with high flow rates of up to 100 ml/min.

A gradient system which made use of flow rate feedback and pneumaticactuation was described by Tsukazaki in U.S. Pat. No. 5,777,213. Thispatent described the advantages for preparative liquid chromatographythat typically operates at flow rates of 100's of ml/minute. This systemmakes use of direct pressurization of a liquid with air which wasdesirable for medicine or food processing. This method would be veryundesirable for capillary HPLC where any bubbles that result fromdissolved gasses will dramatically reduce system performance.Additionally, capillary HPLC is typically run at fairly high fluidpressures (greater than 1000 psi) which would make direct pneumaticcontrol a safety concern.

An additional system, disclosed in Jacques C. LeBlanc, “The StableflowPump—a low-noise and drift-free pump for high performance liquidchromatography” Rev. Sci. Instrum., v62 pp 1642-1646 (1991), wasdeveloped for isocratic HPLC using flow rates between 0.1 and 100ml/minute. This system measures the flow rate after exiting a column anddetection cell. The flow rate was controlled by adjusting thetemperature of a bath that contained a restricting capillary followingthe flowmeter. The desired flow rate was achieved using a feedback loopbetween the flow meter and the temperature bath.

While direct mixing of the fluids is the most robust and useful methodfor generating gradients, accomplishing this in low flow systems isclearly a challenge.

Accordingly, there is a need in the art for a precision flow controlsystem that is capable of delivering fluid at low flow rates in therange of about 1 nanoliter/minute to about 100 microliters/minute andvarying the flow rate in a prescribed manner that is both predictableand reproducible. In addition, it is desirable to have delay volumes of<1 μL and a response time of a few seconds or less.

SUMMARY

The present invention provides a precision flow control system that iscapable of delivering fluid at low flow rates in the range of about 1nanoliter/minute to about 100 microliters/minute and varying the flowrate in a prescribed manner that is both predictable and reproducible.Some embodiments have delay volumes <1 μL and response times of a fewseconds or less.

A precision flow controller system according to the present inventioncomprises a plurality of fluid supplies in fluid communication with afluid outlet so that a plurality of fluids mix before passing throughthe outlet; a pressure source for each fluid applying pressure to therespective fluid; a flowmeter for each fluid measuring the flow rate ofthe respective fluid; and a controller for each pressure source incommunication with the respective flowmeter and the respective pressuresource, wherein the controller compares the respective measured flowrate to a respective desired flow rate and adjusts the respectivepressure source so that the respective fluid flows at the respectivedesired flow rate. Fluid can flow out of the fluid outlet at a flow rateof less than approximately 100 microliters/minute. The desired flowrates do not have to be the same for each fluid and can vary as afunction of time so that the system is suitable to supply fluid to aseparation column.

The flowmeters can be comprised of: a metering capillary having asufficiently long length and sufficiently small inner diameter so thatthe pressure drop across the metering capillary is at least 5% of theinput pressure to the metering capillary; and a pressure sensor formeasuring the pressure drop across the metering capillary. The pressuredrop can be at least 50 psi, for example. The capillary can have aninside diameter of less than approximately 50 microns.

The pressure source can be comprised of: electrokinetic pumps,electrokinetic flow controllers, mechanically activated pumps, andpneumatically activated pumps or any other variable pressure fluidsupply known in the art.

For example, the pressure source can be comprised of: a pneumatic tohydraulic booster in operative fluid communication with the fluidsupply; and a check valve between the fluid supply and the booster sothat fluid flows unidirectionally from the fluid supply to the booster.

The controller can be comprised of; a pneumatic pressure supply inoperative communication with the booster; and a pressure modulatorlocated between the pneumatic pressure supply and the booster, whereinthe pressure modulator controls the amount of pneumatic pressuresupplied to the booster; and a servo-loop controller in communicationwith the respective flowmeter and the pressure modulator, wherein theservo-loop controller compares the measured flow rate to the respectivedesired flow rate and instructs the pressure modulator to adjust thepneumatic pressure supply so that the fluid flows at the desired flowrate.

The system preferably has a time response of less than one second sothat when the measured flow rate does not equal the desired flow rate,the measured flow rate will equal the desired flow rate within onesecond.

In another embodiment, a low flow rate precision flow controllercomprises; a fluid inlet; a fluid outlet in fluid communication with thefluid inlet; a pneumatic pressure supply; a pneumatic to hydraulicbooster located between the fluid inlet and the fluid outlet and inoperative communication with the pneumatic pressure supply, wherein thebooster forces fluid out through the fluid outlet; a pressure modulatorlocated between the pneumatic pressure supply and the booster, whereinthe pressure modulator controls the amount of pneumatic pressuresupplied to the booster; a flowmeter located between the booster and thefluid outlet wherein the flowmeter measures the flow rate of a fluidflowing from the booster to the fluid outlet; and a servo-loop incommunication with the flowmeter and the pressure modulator, wherein theservo-loop compares the measured flow rate to a desired flow rate andinstructs the pressure modulator to adjust the pneumatic pressure supplyso that the fluid flows out of the fluid outlet at the desired flowrate, wherein the desired flow rate is less than approximately 100 microliters/minute. The desired flow rate can also be less than 10microliters/minute. A check valve can be located between the fluid inletand the booster so that the fluid flows unidirectionally from the fluidinlet to the booster.

Preferably, the flow controller has a time response of less than onesecond so that when the measured flow rate does not substantially equalthe desired flow rate, the measured flow rate will substantially equalthe desired flow rate within one second. A method of mixing two fluidsat low flow rates comprising the steps of: applying a pressure source toeach fluid; measuring the flow rate of each fluid before mixing; sendinga desired flow rate for each fluid to the controller; sending themeasured flow rate for each fluid to the controller; and controlling thepressure source of each fluid with the controller so that the measuredflow rates equal the respective desired flow rates. The desired flowrates do not have to be the same for each fluid and can vary as afunction of time. The fluids can flow through a fluid outlet at aconstant flow rate. The flow rate can be measured by a flowmetercomprising: a metering capillary having a sufficiently long length and asufficiently small inner diameter so that the pressure drop across themetering capillary is at least 5% of the input pressure to the meteringcapillary at the desired flow rate; and a pressure sensor for measuringthe pressure drop across the metering capillary. The method can have atime response of less than one second so that when the measured flowrate does not substantially equal the desired flow rate, the measuredflow rate will substantially equal the desired flow rate within onesecond.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 schematically illustrates a precision flow controller system inaccordance with the present invention.

FIG. 2 schematically illustrates the system of FIG. 1 having aninjection valve and a separation column attached to the fluid outlet.

FIG. 3 schematically illustrates a precision flow controller that canmake up part of the system of FIG. 1.

FIG. 4 schematically illustrates a second embodiment of a precision flowcontroller that can make up part of the system of FIG. 1.

FIG. 5 schematically illustrates a third embodiment of a precision flowcontroller that can make up part of the system of FIG. 1.

FIG. 6 schematically illustrates a fourth embodiment of a precision flowcontroller that can make up part of the system of FIG. 1.

FIG. 7 is a detailed illustration of one possible embodiment of thesystem illustrated in FIG. 2.

FIG. 8 is a graph showing the flow rate of a fluid in the controller ofFIG. 6.

FIG. 9 is a graph showing the flow rates of two fluids in the system ofFIG. 7.

FIG. 10 is a graph showing the response time of a pressure modulator ofFIG. 7.

DESCRIPTION

The present invention is directed to a flow controller capable ofproviding a low and controlled flow rate of a liquid. Here ‘low’ meansin the range of about 1 nanoliter per minute to about 100 microlitersper minute. Here ‘controlled’ means the capacity to vary the flow ratein some prescribed manner that is both predictable and reproducible.

A schematic of a basic flow controller system 10 for two fluids is shownin FIG. 1. It includes two variable pressure fluid supplies 12, aflowmeter 14 for each fluid 39, and a single fluid outlet 16. A signalfrom each flowmeter is sent to a controller 18, which in turn adjuststhe pressure of the fluid supplies 12 so that the fluid 39 flows out ofthe fluid outlet 16 at a desired flow rate. The desired flow rate can beless than 100 microliters/minute or less than 10 microliters/minute, forexample.

The fluids 39 are mixed after exiting the flowmeters 14 and beforepassing through the fluid outlet 16. Mixing can occur via diffusion orvia passive or active devices. Ideally, the mixed fluids only need toflow through a minimal volume, 100 nL for example, to the fluid outlet16, the delay volume, so that changes in mixture composition areaccurately represented, with little time delay, in the fluid exitingthrough the fluid outlet.

Preferably, the controller 18 can adjust flow rates of the fluids 39 tocompensate for volumetric changes of the mixture that will affect theflow rate of the mixture. Hence, preferably, the controllers 18 canobtain the physical properties of the fluid upon which the volumedepends, such as composition, temperature and pressure. For example, thecomposition and mixing ratio of both fluids can be input to thecontroller; the flowmeter can measure the pressure; and a thermocouplein communication with the controller can take the temperaturemeasurement. Alternatively, the system can be temperature controlled andthe temperature communicated to the controller.

The flowmeter 14 may be of any type known in the arts including but notlimited to: determining flow rate from measured pressure differenceacross a known flow conductance; a Coriolis flowmeter as disclosed in P.Enoksson, G. Stemme and E. Stemme, “A silicon resonant sensor structurefor Coriolis mass flow measurements,” J. MEMS vol. 6 pp. 119-125 (1997);a thermal mass-flowmeter; a thermal heat tracer as disclosed in U.S.Pat. No. 6,386,050; and an optical flowmeter, for example, a Sagnacinterferometer as disclosed in R. T. de Carvalho and J. Blake,“Slow-flow measurements and fluid dynamics analysis using the Fresneldrag effect, Appl. Opt. vol. 33, pp. 6073-6077 (1994). Preferably theflowmeter 14 provides accurate and precise measurements of flow rates inthe range of 100 μL/min to 10 nL/min. It is further preferable that theflowmeter 14 provide a signal that is continuous over all desired flowrates including fluid flow in both directions. It is further preferablethat the signal bandwidth of the flowmeter 14, i.e. the frequencycorresponding to the minimum time between meaningful readings, is fasterthan one Hertz, and more preferable faster than 10 Hertz.

These objectives can be accomplished by a flowmeter comprising ametering capillary having a sufficiently long length and a sufficientlysmall inner diameter so that the pressure drop across the meteringcapillary is at least 5% of the input pressure to the capillary at thedesired flow rate and a pressure sensors for measuring the pressure dropacross the metering capillary, wherein the input pressure is thepressure of the fluid as it enters the capillary. One or more pressuresensors can be used to measure the pressure drop across the capillarydirectly or by measuring the pressure at both ends of the capillary andsubtracting one pressure measurement from the other. The pressure sensorcan be a pressure transducer. Minimizing the volume and size of thepressure transducers to 5 microliters, for example, allows for rapidresponse of the flowmeter since the compressibility of the fluid and thedeflection of the pressure transducer membrane contribute to the timeresponse.

For example, a pressure drop of about 450 psi through a 10 cm long and10 micron ID capillary indicates a flow rate of about 500 nL/min forwater at room temperature. Similar relations can be determined for otherfluids, geometries, pressure differences, and lengths of tubing usingthe well known Darcy's law for pressure driven flow. Accurate flow ratemeasurements will also require knowledge of the fluid viscosity.

The variable pressure fluid supplies 12 need not be the same and can beof any type known in the arts or developed in the future including butnot limited to: direct electrokinetic pumps, such as those disclosed inU.S. Pat. No. 5,942,093, which is incorporated herein by reference;electrokinetic flow controllers, such as those disclosed in U.S. patentapplication Ser. Nos. 09/942,884 and 10/155,474; electropneumatic pumpswith and without hydraulic amplifiers, such as those described later inthis application; and mechanically actuated pumps. Although many currentdesigns of positive displacement pumps, such as lead-screw driven pumps,do not have the performance to address the precision at the low flowrate ranges, they may be used in active flow rate feedback in futuredesigns. Preferably the variable pressure fluid supplies 12 arecontinuously variable, can provide flow rates in the range of 1nL/minute to 100 μl/minute into back pressures of up to 5000 psi orhigher, and have a response time of seconds or less, thus allowing rapidchanges in flow rates.

Because the flow rate is measured and the measured flow rate is used toadjust the variable pressure fluid supply as opposed to adjusting themechanical displacement of a pump element, e.g., a lead-screw drivenpiston, so that it is proportional to a desired flow rate, the system iscapable of delivering fluid predictably and reproducibly at low flowrates even if there is check valve leakage, pump seal leakage, flexingand creep of mechanical seals, thermal expansion of components andcompression of the working fluid.

Preferably the system has a response time of less than one second sothat when the measured flow rate does not substantially equal thedesired flow rate, the measured flow rate will substantially equal thedesired flow rate within one second, wherein substantially equal meanswithin 5%.

The unique combination of a variable pressure supply with fast timeresponse and the low volume, capillary-based flow meter describedpreviously provides a low flow rate flow controller with excellent timeresponse. The time response of the system can be understood in terms ofthe hydraulic resistances and capacitances. As with an electroniccircuit, the product of these two gives a characteristic time constant.The hydraulic capacitance in the disclosed systems is dominated by thevolumes and compressibility of the fluid, but also includescontributions from sources such as the deflection of the diaphragm inthe pressure transducer.

The capillary flow meter described previously has a reasonably highhydraulic resistance, but a very low fluid volume, allowing rapidresponse. The compressible liquid volume (leading to capacitance) in theillustrated embodiments is on the order of 5 microliters and is due tothe pressure transducer mounting.

For fast flow rate changes in the overall flow controller, the variablepressure source preferably has a rapid time response (i.e., a small timeconstant). In the case of an electrokinetic pump or electrokinetic flowcontroller, the hydraulic resistance may be high, but the compressiblevolume is very low which allows designs with very small time constants.The pneumatic booster pressure supplies later described have largerfluid volumes but the pistons have very low resistance to volumetricchanges—once again allowing very rapid time response.

The combined fluids 39 can be directed to an injection valve 20 and anHPLC separation column 22 as shown in FIG. 2. When this is the case,preferably, the mixture is formed at high pressure and just prior tointroduction to the column 22 thus minimizing the delay volume.Alternatively, or in addition, the combined fluids can be directed toany other components known in the HPLC arts, such as a detector.

A flow controller 24, which comprises the variable pressure fluid supply12, the flowmeter 14, and the controller 18 of the flow controllersystem 10 of FIG. 1, is illustrated in greater detail in FIG. 3. Apneumatic-to-hydraulic booster 26 employs two coupled pistons 28 and 30.A pneumatic pressure supply 32 is coupled to a first piston 28. Theresulting force translates to the fluid 39 in a cylinder 34 on theopposite side of a second piston 30. The gain of the booster 32 isproportional to the ratio of the first piston area to the second pistonarea. This ratio is typically greater than one, but may also be equal toone (direct transfer of pressure with no amplification) or less than onefor lower pressure applications. Thus, the fluid pressure is controlledby varying the pneumatic pressure applied to the first piston 28. Oncethe fluid 39 in the cylinder 34 is expended, the cylinder is refilled bywithdrawing the second piston 30 and pulling fresh fluid from a fluidsupply 38 through a fluid inlet 37 and a check valve 36 into thecylinder 34.

The flow rate as measured by the flowmeter 14 is input to a servo-loopcontroller 40. A desired flow rate is input to the servo-loop controller40 through a set-point input 42. The servo-loop controller 40 thencompares the measured flow rate to the desired flow rate and then, ifthe measured flow rate does not equal the desired flow rate, instructs apressure modulator 44 to adjust the pneumatic pressure to the firstpiston 28 of the booster 26 to achieve the desired liquid flow rate. Thedesired flow rate can be less than 100 microliters/minute or less than10 microliters/minute, for example.

The fluid source 38, the check valve 36 and the pneumatic to hydraulicbooster 26 can make up one of the variable pressure fluid supplies 12 ofFIG. 1. The pneumatic pressure supply 32, the pressure modulator 44, theservo-loop controller 40 and set-point input 42 can make up thecontroller 18 of FIG. 1. The flow controller 24 preferably has a timeresponse of less than one second so that when the measured flow ratedoes not equal the desired flow rate, the measured flow rate equals thedesired flow rate within one second.

The pneumatic-to-hydraulic booster 26 can be of any type known in thearts, including but not limited to a liquid head that uses a dynamicseal on a moving solid rod that displaces liquid, where the rod iscoupled to the shaft of a common pneumatic piston.

The pressure modulator 44 can be any of the means known in the art ofpneumatic control including but not limited to an electro-pneumaticcontroller. Typically in electro-pneumatic controllers, an input currentor voltage produces a command signal to one or more actuators within theelectro-pneumatic controller. This actuator generally acts to increaseor decrease the amount of airflow through the electro-pneumaticcontroller in order to maintain an output pressure proportional to thecommand signal.

The servo-loop controller 40 can be any type known in the art forexample, a PID loop, and can be constructed using discrete analogcircuits, discrete digital circuits, dedicated microprocessors or acomputer, for example.

FIG. 4 shows a variation on the flow controller 24 illustrated in FIG. 3that employs two nested servo loops. An outer servo-loop 40 a comparesthe measured flow rate to the desired flow rate then outputs a pressuresetpoint 43 to an inner servo-loop 40 b. A gas pressure sensor 46measures the pneumatic pressure applied to the first piston 28 of thebooster 26. The measured pneumatic pressure is input to the innerservo-loop 40 b controller as well as the pressure setpoint 43 from theouter servo-loop controller 40 a. If necessary to achieve the desiredflow rate, the inner servo-loop 40 b instructs the pressure modulator 44to adjust the pneumatic pressure applied to the first piston 28 of thebooster 26.

FIG. 5 shows a variant of the flow controller of FIG. 4 wherein one ofthe inner servo-loop 40 b inputs is the measured fluid pressure from afluid pressure sensor 48 located between the booster 26 and theflowmeter 14.

FIG. 6 shows another variant of the device in FIG. 4 wherein theflowmeter 14 comprises a length of capillary tubing 50 having a smalldiameter and a first and a second pressure sensor 48 a and 48 b,respectively, one located on either end of the tubing. The flow rate iscalculated using the measured pressure difference across the known flowconductance of the tubing 50.

The embodiment illustrated in FIG. 6 uses the same inner servo-loop 40 bas the embodiment illustrated in FIG. 4. Alternatively, the firstpressure sensor 48 a of the flowmeter 14 can provide input to the innerservo-loop 40 b.

One possible embodiment of the flow controller system 10 illustrated inFIG. 2 is shown in detail in FIG. 7. The embodiment has two of the flowcontrollers 24 illustrated in FIG. 6, with both flow controllers sharingthe second pressure sensor 48 b. Additionally, the separation column 22leads to a detector 52. In some cases, additional separation columns orother fluidic devices could be inserted after the first separationcolumn 22.

The detector 52 can be any of those known in the HPLC arts, such as: alaser-induced fluorescence detector, an optical absorption detector, arefractive index or electrochemical detector, a mass spectrometer, orNMR spectrometer.

This embodiment can provide a predetermined flow rate of a mixed fluidto the separation column 22 while providing a programmed variation influid composition. The flow rates of a first fluid 39 a and a secondfluid 39 b from their respective sources 38 can be independentlymeasured and servo-controlled to meter the independent flowsproportional to the respective set-point inputs. The programmedvariation in fluid composition can be in the form of a series of stepchanges, a continuous ramp, i.e. a gradient, or any of the other formsknown in the separation arts. In other embodiments, attendant flowcontrollers and servo loops can be combined to provide for morecomplicated or broad ranging fluid composition variations.

Thus, two or more flow controllers 24 can be combined to deliver fast,accurate, and reproducible gradients for use in microscale separationsthat require extremely low flow rates. In this system, all of the flowgoes directly into the separation column 22 and no excess fluid isshunted to waste as is commonly employed in systems with flow splitting.Additionally, the attainable low flow rates enable the system to supplyan eluant directly into a mass spectrometer. Flow controllers embodyingthe invention can also be run in parallel from common sources of fluidsto perform multiple separations in parallel.

Embodiments of the invention provide a liquid flow rate that isrelatively free of pressure pulsations. Embodiments of the invention canbe configured to provide a controlled flow rate of liquid at pressureranging from about one atmosphere to over 10,000 psi. Applicationsinclude but are not limited to: gradient HPLC, fluid delivery to a massspectrometer, flow injection analysis, drug delivery, and supply ofliquid reactants to a chemical reactor.

EXAMPLES

FIG. 8 shows the measured flow rate from the system described in FIG. 6,where the setpoint was set to a constant value of 2700 nL/min over 50minutes. In this system, the pressure modulator 44 was anelectro-pneumatic controller Control Air 900-EHD, thepneumatic-to-hydraulic booster 26 was Haskel MS-36, and the pump fluidwas deionized water. The pressure sensors 48 a and 48 b were two EntranEPX transducers. The transducers were on either side of a 10 cm lengthof a 10 μm ID capillary tube 50. The pressure drop across the capillarytube was about 100 psi. The flow rate was calculated by amicroprocessor. The resulting metered flow of liquid was accurate within0.02% of its setpoint over the duration of the test, or a flow rateaccuracy of 0.56 nL/min RMS.

The data shown in FIG. 9 were generated using the system shown in FIG.7. The flow profile was a 20 minute gradient delivery of the first andsecond fluids, 39 a and 39 b, respectively, water and acetonitrile,respectively, with a one minute period of constant flow at the beginningand end of the test. Each flow controller was programmed to vary itsflow rate over a range of 300 to 100 nL/min. The two streams of fluidwere mixed in concentrations ranging from 25% to 75% over the durationof the test. The mixture ratio varied linearly over time so that thesummed flows were maintained at a constant flow rate of 400 nL/min intothe column. The measured flow rate from each controller indicatesaccuracy within 0.28 nL/min RMS of the setpoint over the full range ofthe test conditions.

FIG. 10 shows the response time of the pressure modulators of FIG. 7.The response time is demonstrated to be less than one second so thatwhen the desired flow rate does not equal the measured flow rate, themeasured flow rate will equal the desired flow rate within one second.Fast response changes in flow rate, as illustrated in FIG. 10, can beused in a number of analytical methods to conduct stopped flow orpeak-parking to increase integration time and improve detectionsensitivity.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. For example, three or more flow controllers can be used in asingle flow controller system. Therefore, the spirit and scope of theappended claims should not be limited to the description of thepreferred versions contained herein.

All features disclosed in the specification, including the claims,abstracts, and drawings, and all the steps in any method or processdisclosed, may be combined in any combination, except combinations whereat least some of such features and/or steps are mutually exclusive. Eachfeature disclosed in the specification, including the claims, abstract,and drawings, can be replaced by alternative features serving the same,equivalent or similar purpose, unless expressly stated otherwise. Thus,unless expressly stated otherwise, each feature disclosed is one exampleonly of a generic series of equivalent or similar features.

Any element in a claim that does not explicitly state “means” forperforming a specified function or “step” for performing a specifiedfunction should not be interpreted as a “means” for “step” clause asspecified in 35 U.S.C. §112.

What is claimed is:
 1. A low flow rate precision flow controller systemcomprising: (a) a plurality of continuously variable pressure fluidsupplies delivering a plurality of fluids; (b) a single fluid outletbeing in fluid communication with the plurality of fluid supplies, theplurality of fluids being mixed before exiting through the outlet; (c) aplurality of pressure sources corresponding to the plurality of fluids,each pressure source applying pressure to each fluid; (d) a plurality ofcapillary tube thermal mass flowmeters, each flowmeter detecting theflow rate of each fluid of the corresponding plurality of fluids; and(e) a controller for electronic flow control in communication with therespective flowmeter and the respective pressure source, wherein thecontroller compares the respective measured flow rate to a respectivedesired flow rate and adjusts the respective pressure source so that therespective fluid flows at the respective desired flow rate, wherein thefluids flow out of the fluid outlet at a flow rate of less thanapproximately 100 microliters/minute.
 2. The system of claim 1, whereinthe desired flow rates are different in values for each fluid.
 3. Thesystem of claim 1, wherein the desired flow rates are less than 10microliters/minute.
 4. The system of claim 1, wherein the desired flowrates of the fluids vary as a function of time.
 5. The system of claim1, wherein the mixed fluids flow through the fluid outlet at asubstantially constant flow rate.
 6. The system of claim 1, furthercomprising a separation column in fluid communication with the fluidoutlet.
 7. The system of claim 6, further comprising a detector in fluidcommunication with an outlet end of the separation column.
 8. The systemof claim 1, wherein each flowmeter comprises a metering capillary havingan inside diameter of less than 50 microns.
 9. The system of claim 1,wherein the mixed fluid exiting the flowmeter flows through a minimalvolume of 100 nL before exiting the fluid outlet.
 10. The system ofclaim 1, wherein the pressure sources are mechanical pumps.
 11. Thesystem of claim 1, wherein at least one pressure source comprises anelectrokinetic pump.
 12. The system of claim 1, wherein at least onepressure source comprises an electrokinetic flow controller.
 13. Thesystem of claim 1, wherein at least one pressure source comprises apneumatically activated pump.
 14. The system of claim 1, wherein atleast one pressure source comprises a mechanically activated pump. 15.The system of claim 1, wherein the system has a time response of lessthan one second so that when the measured flow rate does notsubstantially equal the desired flow rate, the measured flow rate willsubstantially equal the desired flow rate within one second.
 16. A lowflow rate precision flow controller system comprising: (a) a pluralityof fluid supplies in fluid communication with a fluid outlet so that aplurality of fluids mix before flowing through the outlet; (b) amechanical pump for each fluid applying pressure to the respectivefluid; (c) a capillary-tube thermal mass flowmeter for each fluid formeasuring the flow rate of the respective fluids; (d) a thermocouple incommunication with the capillary-tube thermal mass flowmeter for takinga temperature measurement; and (e) at least one controller for eachmechanical pump in communication with the respective flowmeter viarespective thermocouple and the respective pressure source, wherein theat least one controller compares the respective measured flow rate to arespective desired flow rate and adjusts the respective mechanical pumpso that the respective fluid flows at the respective desired flow rate,wherein the fluids flow out of the fluid outlet at a flow rate of lessthan approximately 100 microliters/minute.